Fluid Processing Device for Oligonucleotide Synthesis and Analysis

ABSTRACT

The present teachings provide a fluid processing device adapted to produce different oligomers in a plurality of respective reaction sites. The fluid processing device can comprise a first manifold for delivering reactants to the plurality of reaction sites, and a second manifold for removing waste from, and optionally delivering wash fluid to, the plurality of reaction sites. Surface tension control valves can be disposed in fluid communication with the first manifold and can selectively allow reactants and/or fluids into the reaction sites. A method of making oligonucleotides is also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. patent application Ser. No.11/092,180, filed May 29, 2005, the entire content of which isincorporated herein by reference.

FIELD

The present teachings relate to fluid processing devices and methods forusing the same.

BACKGROUND

One of the challenges encountered in fluid processing devices,particularly devices designed for high throughput operations, is how toeffectively control fluid flow. It is especially difficult toindividually and independently control fluid flow in thousands ofmicro-channels without resorting to the fabrication of sophisticatedvalving systems which can make microfluidic devices very expensive. Adevice and method for controlling fluid flow in a microfluidic system isdesirable.

SUMMARY

According to various embodiments, a fluid processing device is providedthat includes a plurality of reaction sites, a first manifold in fluidcommunication with each of the reaction sites, a second manifold influid communication with each of the reaction sites, and at least onesurface tension control valve positioned in at least one channel betweenthe first manifold and at least one of the reaction sites. The reactionsites can each comprise support structures, for example beads, or aninner surface, suitable for the attachment of oligomers or oligomerprecursors thereto. According to various embodiments, the fluidprocessing device can comprise a plurality of surface tension controlvalves each in fluid communication with the first manifold and one ormore of the reaction sites.

According to various embodiments, the fluid processing device cancomprise reactants and/or reaction components capable of producing anoligomer in at least one of the reaction sites, or a system thatincludes sources of reactants and/or reaction components.

According to various embodiments, a system is provided that can comprisea fluid processing device as described herein, and an electromagneticradiation source capable of emitting electromagnetic radiation anddirecting the radiation toward one or more surface tension controlvalves in the device. Alternatively or additionally, the system cancomprise other valve-actuating devices besides an electromagneticradiation source. Exemplary actuators can comprise heaters adapted todirect heat toward one or more surface tension control valves, or anelectrical source adapted to supply an electrical signal to one or moresurface tension control valves. By controlling the one or more surfacetension control valves, the systems described herein can be used indirecting the flow of reaction components in an order useful forcarrying out an oligonucleotide synthesis reaction within one or more ofthe plurality of reaction sites.

According to various embodiments, a system is provided that comprises anelectromagnetic radiation source or other actuating source, a reflectivedevice, a pump, and a thermocycler. The system can be adapted so thatthe reflective device can direct electromagnetic radiation emitted fromthe electromagnetic radiation source toward the one or more surfacetension control valves to selectively open or close the respective oneor more surface tension control valves. The pump can be adapted to addor remove materials from the channels and reaction sites. Thethermocycler can be adapted to control the temperature of the reactionsites, for example, to promote an isothermal or thermally cycled nucleicacid sequence amplification and/or detection assay. The system cancomprise one or more control units to control the actuating source, tocontrol the reflective device, to control the pump, and/or to controlthe thermocycler.

According to various embodiments, a method is provided for synthesizingoligonucleotides or other chemical structures, from component buildingblocks. The method can comprise, for example, introducing a firstmonomer into a first fluid distribution manifold of a fluid processingdevice; opening at least one surface tension control valve in fluidcommunication with both the first fluid distribution manifold and atleast one respective reaction site, to form an open surface tensioncontrol valve; moving the first monomer from the first manifold, throughthe at least one open surface tension control valve, and into the atleast one respective reaction site; and attaching the first monomer to afirst structure in the at least one respective reaction site to form anextended structure. The first monomer can be, for example, a nucleotide,a nucleotide base, a nucleotide analog, a protected chemical buildingblock, or another monomeric building block, unit, or structure that canbond with and extend off of a support or precursor structure. The firstmonomer can be a protected first monomer, the extended structure can bea protected extended structure, and the method can further comprise:washing the at least one respective reaction site subsequent to theattaching; closing the at least one surface tension control valve;introducing a deprotecting agent into the first manifold; opening the atleast one surface tension control valve to form at least one reopenedsurface tension control valve; moving the deprotecting agent from thefirst manifold, through the at least one reopened surface tensioncontrol valve, and into the at least one respective reaction site; anddeprotecting the extended protected structure to form a deprotectedextended structure. An additional monomer can then be added to thedeprotected extended structure and the process can be repeated.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present teachings are exemplified by theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and as would be known or recognized by thoseof ordinary skill in the art given the present teachings. In thedrawings:

FIG. 1 is a cross-sectional view through a portion of a device accordingto various embodiments and including a surface tension control valve;

FIG. 2A is a cross-sectional view through a portion of a deviceaccording to various embodiments and including a surface tension controlvalve in a closed state;

FIG. 2B is the same cross-sectional view shown in FIG. 2A, but after thesurface tension control valve has been opened;

FIG. 3A is a side view of a surface tension control valve showing inphantom the effect that actuation of a surface tension control devicehas on the shape of a drop of water;

FIG. 3B is a side view of a surface tension control device showing inphantom the effect that actuation of the device has on the shape of adrop of water;

FIG. 3C is a partial cross-sectional view of a surface tension controldevice showing the direction of movement of a drop of water, resultingfrom actuation of the valve;

FIG. 4A is a cross-sectional view through a portion of a deviceaccording to various embodiments and showing a light-activated surfacetension control valve in a closed state;

FIG. 4B is the same cross-sectional view as shown in FIG. 4A, butwherein the light-activated surface tension control valve is in an openstate;

FIG. 4C is the same cross-sectional view as shown in FIG. 4A, butwherein the light-activated surface tension control valve is in an openstate and liquid has passed through the valve;

FIG. 4D is the same cross-sectional view as shown in FIG. 4A, butwherein the valve is in a closed state after liquid has passed throughthe valve;

FIG. 5 is a top plan view of a portion of a fluid processing deviceaccording to various embodiments;

FIG. 6 is a top plan close-up view of a portion of a fluid processingdevice according to various embodiments;

FIG. 7 is a perspective view of a system for processing a fluidprocessing device according to various embodiments;

FIG. 8 is a perspective view of a system for processing a fluidprocessing device according to various embodiments; and

FIG. 9 is a perspective view of yet another system for processing afluid processing device according to various embodiments.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are intended to provide an explanation of various embodiments of thepresent teachings.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENT

According to various embodiments, a fluid processing device is providedthat can be used to manipulate the delivery of reactants or reactioncomponents to a reaction site to enable the production of one or morecompounds comprising multiple building blocks, for example, one or moredesired oligomers or one or more desired oligonucleotides. Oligomers asdefined herein can include polymers of amino acids, polymers of sugars,polymers of nucleotide bases, polymers of nucleotide analogs, and/orpolymers of other nucleotide monomeric units herein referred to asnucleotides.

According to various embodiments, the device described herein can beuseful in carrying out chemical compound synthesis methods usingbuilding blocks, exemplified herein with oligonucleotide synthesismethods. These methods can comprise, for example, variousoligonucleotide extension reactions, protecting and/or deprotectingreactions, capping reactions, washing steps, cleaving reactions, and thelike. Exemplary oligonucleotide synthesis reactions can include thosedescribed, for example, in U.S. patent application Ser. No. 10/891,650,filed Jul. 15, 2004, which is incorporated herein in its entirety byreference.

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or substituted or unsubstituted aromaticrings. In certain embodiments, the aromatic ring or rings contain atleast one nitrogen atom. In certain embodiments, the nucleotide base iscapable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with anappropriately complementary nucleotide base. Exemplary nucleotide basesand analogs thereof include, but are not limited to, naturally occurringnucleotide bases, adenine, guanine, cytosine, 6 methyl-cytosine, uracil,thymine, and analogs of the naturally occurring nucleotide bases, e.g.,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, N6-Δ2-isopentenyladenine (6iA),N6-Δ2-isopentenyl-2-methylthioadenine (2 ms6iA), N2-dimethylguanine(dmG), 7-methylguanine (7mG), inosine, nebularine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O6-methylguanine, N6-methyladenine,O4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,pyrazolo[3,4-D]pyrimidines (see, e.g., U.S. Pat. Nos. 6,143,877 and6,127,121 and PCT published application WO 01/38584), ethenoadenine,indoles such as nitroindole and 4-methylindole, and pyrroles such asnitropyrrole. Certain exemplary nucleotide bases can be found, e.g., inFasman, 1989, Practical Handbook of Biochemistry and Molecular Biology,pp. 385-394, CRC Press, Boca Raton, Fla., and the references citedtherein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR2 or halogen groups, where each Ris independently H, C1-C6 alkyl, or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-anomeric nucleotides, 1′-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). Exemplary LNA sugar analogs within a polynucleotideinclude, but are not limited to, the structures:

where B is any nucleotide base.

Modifications at the 2′- or 3′-position of ribose include, but are notlimited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino,alkylamino, fluoro, chloro, and bromo. Nucleotides include, but are notlimited to, the natural D optical isomer, as well as the L opticalisomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res. 21:4159-65;Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) NucleicAcids Symposium Ser. No. 29:69-70). When the nucleotide base is purine,e.g. A or G, the ribose sugar is attached to the N9-position of thenucleotide base. When the nucleotide base is pyrimidine, e.g. C, T or U,the pentose sugar is attached to the N1-position of the nucleotide base,except for pseudouridines, in which the pentose sugar is attached to theC5 position of the uracil nucleotide base (see, e.g., Kornberg andBaker, (1992) DNA Replication, 2nd Ed., Freeman, San Francisco, Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where α is an integer from 0 to 4. In certain embodiments, α is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with atriphosphate ester group at the 5′ position, and are sometimes denotedas “NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group may includesulfur substitutions for the various oxygens, e.g. -thio-nucleotide5′-triphosphates. For a review of nucleotide chemistry, see: Shabarova,Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH,New York, 1994.

The term “nucleotide analog”, as used herein, refers to embodiments inwhich the pentose sugar and/or the nucleotide base and/or one or more ofthe phosphate esters of a nucleotide may be replaced with its respectiveanalog. In certain embodiments, exemplary pentose sugar analogs arethose described above. In certain embodiments, the nucleotide analogshave a nucleotide base analog as described above. In certainembodiments, exemplary phosphate ester analogs include, but are notlimited to, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, and the like,and may include associated counterions.

Also included within the definition of “nucleotide analog” arenucleotide analog monomers that can be polymerized into polynucleotideanalogs in which the DNA/RNA phosphate ester and/or sugar phosphateester backbone is replaced with a different type of internucleotidelinkage. Exemplary polynucleotide analogs include, but are not limitedto, peptide nucleic acids, in which the sugar phosphate backbone of thepolynucleotide is replaced by a peptide backbone. Also included areintercalating nucleic acids (INAs, as described in Christensen andPedersen, 2002), and AEGIS bases (Eragen, U.S. Pat. No. 5,432,272).

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably and mean single-stranded ordouble-stranded polymers of nucleotide monomers, including2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked byinternucleotide phosphodiester bond linkages, or internucleotideanalogs, and associated counter ions, e.g., H+, NH4+, trialkylammonium,Mg2+, N+ and the like. A nucleic acid can be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. The nucleotide monomer units can comprise any of thenucleotides described herein, including, but not limited to, naturallyoccurring nucleotides and nucleotide analogs. Nucleic acids typicallyrange in size from a few monomeric units, e.g. 5-40 when they aresometimes referred to in the art as oligonucleotides, to severalthousands of monomeric nucleotide units. Unless denoted otherwise,whenever a nucleic acid sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine or an analog thereof, “C” denotesdeoxycytidine or an analog thereof, “G” denotes deoxyguanosine or ananalog thereof, and “T” denotes thymidine or an analog thereof, unlessotherwise noted.

Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA,mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained fromsubcellular organelles such as mitochondria or chloroplasts, and nucleicacid obtained from microorganisms or DNA or RNA viruses that may bepresent on or in a biological sample.

Nucleic acids may be composed of a single type of sugar moiety, e.g., asin the case of RNA and DNA, or mixtures of different sugar moieties,e.g., as in the case of RNA/DNA chimeras. In certain embodiments,nucleic acids are ribopolynucleotides and 2′-deoxyribopolynucleotidesaccording to the structural formulae below:

wherein each B is independently the base moiety of a nucleotide, e.g., apurine, a 7-deazapurine, a pyrimidine, or an analog nucleotide; each mdefines the length of the respective nucleic acid and can range fromzero to thousands, tens of thousands, or even more; each R isindependently selected from the group comprising hydrogen, halogen, —R″,—OR″, and —NR″R″, where each R″ is independently (C1-C6) alkyl or(C5-C14) aryl, or two adjacent Rs are taken together to form a bond suchthat the ribose sugar is 2′,3′-didehydroribose; and each R′ isindependently hydroxyl or

where α is zero, one or two.

In certain embodiments of the ribopolynucleotides and2′-deoxyribopolynucleotides illustrated above, the nucleotide bases Bare covalently attached to the C1′ carbon of the sugar moiety aspreviously described.

The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” canalso include nucleic acid analogs, polynucleotide analogs, andoligonucleotide analogs. The terms “nucleic acid analog”,“polynucleotide analog” and “oligonucleotide analog” are usedinterchangeably and, as used herein, refer to a nucleic acid thatcontains at least one nucleotide analog and/or at least one phosphateester analog and/or at least one pentose sugar analog. Also includedwithin the definition of nucleic acid analogs are nucleic acids in whichthe phosphate ester and/or sugar phosphate ester linkages are replacedwith other types of linkages, such as N-(2-aminoethyl)-glycine amidesand other amides (see, e.g., Nielsen et al., 1991, Science 254:1497-1500; WO 92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No.5,698,685); morpholinos (see, e.g., U.S. Pat. No. 5,698,685; U.S. Pat.No. 5,378,841; U.S. Pat. No. 5,185,144); carbamates (see, e.g., Stirchak& Summerton, 1987, J. Org. Chem. 52: 4202); methylene(methylimino) (see,e.g., Vasseur et al., 1992, J. Am. Chem. Soc. 114: 4006);3′-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);2-aminoethylglycine, commonly referred to as PNA (see, e.g., Buchardt,WO 92/20702; Nielsen (1991) Science 254:1497-1500); and others (see,e.g., U.S. Pat. No. 5,817,781; Frier & Altman, 1997, Nucl. Acids Res.25:4429 and the references cited therein). Phosphate ester analogsinclude, but are not limited to, (i) C1C4 alkylphosphonate, e.g.methylphosphonate; (ii) phosphoramidate; (iii) C1C6alkyl-phosphotriester; (iv) phosphorothioate; and (v)phosphorodithioate.

The surface tension control valve that can be used according to variousembodiments are herein exemplified by an implementation represented by achannel having an inner surface that is hydrophobic in the absence ofilluminating radiation. According to various embodiments, the surfacetension control valve can exploit the fact that under certaincircumstances the contact angle for a liquid of interest, or its surfacetension, changes and that change can in-turn trigger a movement of theliquid. Such circumstances can comprise an applied electric field(electrowetting), an applied electric field and light(optoelectrowetting), an applied local increase of temperature (thermocapillary effect), and the like. The liquid can be a liquid sample, forexample, a biological sample in water or a biological sample in anaqueous medium. If the liquid is a biological sample, it can comprise,for example, any of the nucleotides, nucleotide bases, and/or nucleotideanalogs described herein.

Exemplary surface tension control valves can comprise the microfluidicelectrowetting control devices described in U.S. Patent ApplicationPublication No. US 2004/0231987 A1, published Nov. 25, 2004; theelectrostatic actuators for microfluidics described in U.S. PatentApplication Publication No. US 2002/0043463 A1, published Apr. 18, 2002;the micropump device as described in U.S. Patent Application PublicationNo. US 2002/0114715 A1, published Aug. 22, 2002; the electrowettingmicrofluidic control device described in U.S. Patent ApplicationPublication No. US 2003/0164295 A1, published Sep. 4, 2003; the controldevices described in U.S. Patent Application Publication No. US2002/0168671 A1, published Nov. 14, 2002; the optical microfluidicdevices described in U.S. Patent Application Publication No. US2003/0047688 A1; and the injecting devices as described in U.S. PatentApplication Publication No. US 2003/0082081 A1; all of which areincorporated herein in their entireties by reference.

The present teachings are further exemplified herein with reference tothe attached drawings. According to various embodiments, and asillustrated in FIG. 1, the fluid processing device 100 can comprise asurface tension control valve 108 disposed in a valve channel 106 thatis in fluid communication with a supply channel 104 and a reactionregion 102. In the embodiment shown, the surface tension control valvecomprises a channel with an initially or normally hydrophobic surface.The surface tension control valve is adapted to change the contact angleand wetting of a liquid disposed therein with respect to the innersurface of the valve channel 106. This change can trigger the movementof a liquid through the valve channel 106. As discussed above, examplesof the mechanism that can be used to trigger the movement can includethe application of an electric field as with electrowetting, theapplication of an electric field and light as with optoelectrowetting,the application of a local increase in temperature, and the like.

According to various embodiments, and as illustrated in FIG. 2A, aportion of a fluid processing device is shown comprising a first conduit204 separated from a second conduit 206 by a surface tension controlvalve 208. The pressure created by the surface tension in the surfacetension control valve 208 can be sufficient to prevent a liquid 202, forexample, an aqueous biological sample, from entering a second conduit206. If the pressure difference across the surface tension control valveexceeds a certain threshold pressure, the resistance to the flow due tothe hydrophobic properties of the valve can be overcome, and the liquidcan flow through the valve. Likewise, if the pressure of the sampleliquid is maintained below the threshold pressure, the valve will holdback the liquid sample and prevent flow into channel 206. According tovarious embodiments, by changing the surface tension of the valve fromhaving a hydrophobic property to having a hydrophilic property, liquidmovement through the valve can be regulated, even at pressure below thethreshold pressure described above.

FIG. 2B illustrates the same device as shown in FIG. 2A but wherein thesurface tension of the surface tension control valve 208 has beenchanged to be made hydrophilic, thus enabling liquid 210 to pass throughthe valve 208 and into the second conduit 206. Changing the surfacetension of the valve can be accomplished by a variety of mechanism asdescribed herein.

According to various embodiments, and as illustrated in FIG. 3A, thesurface tension control valve can comprise a layered structure 300capable of changing the surface tension of a surface. The layeredstructure 300 can comprise a first electrode-containing layer 302positioned adjacent a second insulating layer 304. When the structure300 is connected to a power source 312 through electrical leads 310 and311, a change in surface tension can be effected by application of anelectrical signal to the electrical leads 310 and 311. As a result ofsuch a signal, the valve can change the overall shape of a liquiddroplet from a first shape 308 having a greater contact angle, to asecond shape 306 having a lesser contact angle, by creating a differencein electrical potential between the liquid and the electrode. Byincreasing or decreasing the power of the electrical signal, the shapeof the droplet can be changed to take any of a variety of forms, asillustrated in FIGS. 3A and 3B.

According to various embodiments, and as illustrated in FIG. 3B, thefluid processing device can comprise a surface tension control valvethat comprises a layered structure 316. The layered structure 316 cancomprise a photoconductive layer 318, an electrode-containing layer 320,and an insulating layer 322. A power supply 330 can be connected throughleads 328 and 329 to a liquid bead. Applying current will change theshape of the liquid droplet from the shape depicted in 326 to the shapedepicted in 324. In other words, when illuminated by light, thephotoconductive layer of the surface tension control valve can changelocally and significantly in conductivity and, as a result, the surfaceof the insulating layer 322 that contacts the liquid droplet can be madehydrophilic or more hydrophilic. The contact angle or wetting of theliquid with respect to the surface can thus be changed and the liquidcan accordingly be propagated in a certain direction.

According to various embodiments, and as illustrated in FIG. 3C, lightbeams 358 can change the electrical resistance of a photoconductivelayer 348 in a light-activated valve 340, allowing electrical current toflow through one or more electrodes 350. Electrical current flow changesthe surface tension of the liquid in parts of the liquid droplet 360allowing liquid droplet 360 to flow toward the light beams 358.

According to various embodiments, and as illustrated in FIGS. 4A-4D, afluid processing device 400 can comprise a first conduit 402 separatedfrom a second conduit 404 by a light-activated surface tension controlvalve 406. When an area 410 including the valve 406 is illuminated bybeams of light, the surface tension of the valve 406 changes and enablesa liquid droplet 414 (FIG. 4D) to be separated from liquid 412 presentin the first conduit 402. Conduit 408 can function to relieve airpressure differences caused by the movement of drop 414 into the secondchannel 404. Conduit 408 can include a first open end 409 in fluidcommunication with the interior of the valve 406, and a second open end411 in fluid communication with the second conduit 404.

According to various embodiments and as depicted in FIGS. 4A-4D, asurface tension control valve separating a first liquid-containingconduit and a second conduit can normally or originally be in a closedstate in the absence of illuminating radiation. If a light beamilluminates the surface tension control valve where the liquid contactsthe valve, the valve surface can be made hydrophilic, enabling theliquid to move into the valve. If the beam of light is then movedtowards the second conduit, the beam can be followed by the liquid asthe localized surface tension of the valve is changed. Once the lightbeam moves past the valve surface such that part of the valve is nolonger illuminated. The localized valve surface will again becomehydrophobic and will be closed. A volume of liquid can thus be taken-upfrom and broken away from the liquid in the first channel. Uponcontinued movement of the light beam followed by switching off thelight, the remaining liquid in the valve can be moved into the secondchannel.

According to various embodiments, and as illustrated in FIG. 5, a fluidprocessing device 500 can comprise a first manifold 503 in fluidcommunication with a supply conduit 504. The first manifold 503 can bein fluid communication with several feeder conduits 506. A plurality ofreaction sites 510 can be in fluid communication with a respectivefeeder channel 506. Surface tension control valves 508 can be disposedin one or more of the feeder channels 506 adjacent each reaction site510. A second manifold 517 is in fluid communication with a conduit 516and a plurality of feeder conduits 514. Each feeder conduit 514 is influid communication with a respective plurality of the reaction sites510, for example, on opposite sides of the respective reaction regionsrelative to the respective feeder channels 506.

The fluid processing device 500 can be disposed in or upon a chip orcard 502. The chip or card 502 can comprise glass, silicon, plastic,polycarbonate, polypropylene, polymers of cyclic olefins, copolymers,combinations thereof, and the like. The chip or card 502 can be moldedwith features and enclosed by one or more cover films or layers. Thereaction regions 510 can be any suitable shape, for example,well-shaped.

The conduits and reaction sites can have any of a variety of dimensions.At least one feature can have at least one dimension of five mm or less,for example, one mm or less, or 500 microns or less. Conduit depths andwidths can be equivalent or different from one another. Differentchannel aspect ratios can be used. According to various embodiments,channels can be dimensioned to permit manipulation of fluids bycapillary action, and to promote or induce capillary fluid flow. Theconduits can have various cross-sectional shapes, including, forexample, a square cross-section, a rectangular cross-section, a circularcross-section, a U-shaped cross-section, a V-shaped cross-section, or acombination thereof.

If a conduit has an inner surface that contains both hydrophobic andhydrophilic portions, some additional force or pressure can be requiredto push the liquid through the hydrophobic part of the channel ascompared movement through a hydrophilic portion of the same channel.

According to various embodiments, and as illustrated in FIG. 6, a fluidprocessing device 600 can comprise a substrate 601, and a first manifold604 including a main conduit 606 and several feeder conduits 620, 622,and 624, wherein each feeder conduit comprises a respective surfacetension control valve. The feeder conduits 620, 622, 624 can be in fluidcommunication with reaction sites 614, 616, and 618, respectively.Reaction sites 616 and 618 can be fluidically connected to one anotherby a conduit 626 that comprises a surface tension control valve. Theconduit 626 can be directly between the reaction sites 616 and 618.Similarly, reaction sites 614 and 616 can be fluidically connected by aconduit 628 having a surface tension control valve. Controlling theopening and closing of one or more of the surface tension control valves620, 622, 624 can enable the selective production of a differentoligomer in each respective reaction site 614, 616, 618. A secondmanifold 606 comprising a main conduit 608 and several feeder conduits610, 612, 614 can be in fluid communication with the reaction sites 614,616, 618, respectively. The second manifold 606 can be used to carryaway reactants, non-reactive reaction components, and/or wash fluids,from the reaction regions. The second manifold 606 can alternatively, oradditionally, be used to supply the reaction sites 614, 616, 618 withone or more reactants, non-reactive reaction components, and washfluids. Through combinations of supply and wash steps and surfacetension control valve opening and closing steps, differentoligonucleotides can simultaneously be synthesized in the differentreaction sites of the device, as described in more detail below.

According to various embodiments, and as illustrated in FIG. 7, a fluidprocessing system 700 can comprise a processing device, for example, acomputer 702. The computer can be electrically connected, for example,through wires or through a wireless connection, to a suitableelectromagnetic radiation source 706 that is capable of sufficientlyilluminating a surface tension control valve to cause a change in thehydrophobic/hydrophilic properties of the valve. The electromagneticradiation source 706 can comprise a laser, an ultraviolet light source,an infrared source, an incandescent bulb, a fluorescent bulb, alight-emitting diode (LED), an array of LEDs, combinations thereof, andthe like.

According to various embodiments, the fluid processing system 700 cancomprise an apparatus 704 for directing the electromagnetic radiationtoward a plurality of separate surface tension control valvesincorporated in a fluid processing device, for example, in a card orchip 710. The apparatus 704 can include an electromagnetic radiationreflective device such as one or more minors. The apparatus 704 cancomprise a plurality of independently-moveable, computer controllable,micro-minors 705, as shown. The fluid processing system 700 can furthercomprise one or more lenses 708, for focusing the electromagneticradiation reflected by the micro-mirrors 705 toward the fluid processingdevice 710. Pumps 712, and 714, can be fluidically connected to thefluid processing device 710, for example, to one or more manifolds inthe device, and operatively connected to the computer 702. Operativelyconnected can be defined as electrically connected, mechanicallyconnected, fluidically connected, combinations thereof, and the like.The pumps 712, 714, can be used to control, at least in-part, the flowof fluids to and/or from the fluid processing device 710.

According to various embodiments, and as illustrated in FIG. 8, a fluidprocessing system 800 is provided that can include a single mirror, forexample, a galvo-mirror, controlled by a computer 801, to directelectromagnetic radiation from an electromagnetic radiation source 802through one or more lenses 804 toward light-activated surface tensioncontrol vales in a fluid processing device 806.

According to various embodiments, and as illustrated in FIG. 9, a fluidprocessing system 900 can include a rotatable carousel 902 having a topsurface. Disposed upon the top surface of the carousel 902 can be aplurality of fluid processing devices 904. Each device 904 can comprisea first manifold, a second manifold, a plurality of reaction regions,and a plurality of surface tension control valves, as described above.The carousel 902 can rotate so as to position each device 904 above aheater 906, and adjacent two or more pumps or pumping blocks 908, 910.The pumping blocks 908, 910 can be any suitable pumping devices formoving reagents, or can be pumping systems capable of independentlyaddressing and pumping a number of different reagents present inside theblock itself or in fluid communication with the blocks. Reagents thatcan be pumped into and out of the devices by the pumping blocks 908 and910 can include nucleotides, nucleosides, nucleotide analogs, adenine,cytosine, guanine, thymine, uracil, protected versions thereof,deprotecting reagents, acids, capping reagents, wash fluids, orcombinations thereof.

A detection block 912 comprising an electromagnetic radiation source andan imaging system can be disposed above the carousel 902. The detectionblock 912 can comprise an electromagnetic radiation source capable ofselectively opening one or more surface tension control valves of anunderlying device 904. The detection block 912 can also comprise animaging system capable of recording images of, or viewing, taggedmolecules, for example, fluorescently tagged molecules. The imagingsystem can include, for example, an analog camera, a film camera, adigital camera, a CCD, or a combination thereof. The fluid processingsystem 900 can include a drive unit 905 and a control unit 914. Thecontrol unit 914 can be operatively connected to the optical block 912,the pumping blocks 908, 910, the heater 906, the carousel 902, and/orthe drive unit 905. Operatively connected can be defined as electricallyconnected, mechanically connected, fluidically connected, combinationsthereof, and the like.

According to various embodiments, a method of synthesizing oligomers,for example, oligonucleotides, is provided for which traditionalphosphoramidite chemistry can be used. The method can be used to createa plurality of identical oligomers in each reaction site or to create adifferent oligomer in each respective reaction site. The method caninclude providing a fluid processing device as described herein, forexample, that includes one or more reaction sites each including aninner surface, a first manifold in fluid communication with the one ormore reaction sites, a second manifold in fluid communication with theone or more reaction sites, and one or more surface tension controlvalves disposed in the first manifold.

The method can include introducing a first protected monomer into thefirst manifold, whereby the first protected monomer can be selectivelyintroduced, through the one or more surface tension control valves, intothe one or more reaction sites, depending upon how many surface tensioncontrol valves are activated to become open. The first protected monomercan then be attached to a structure or precursor in each reaction site,or can be attached directly to the inner surface of each reaction site.The attachment forms an extended structure. Excess first protectedmonomer can then be drawn out of the one or more reaction sites andthrough the second manifold, for example, by using a pumping block ordevice to create a negative pressure differential. At the same time, orsubsequently, a wash fluid from the first manifold or second manifoldcan be forced into, or drawn through and away from, the one or morereaction sites that had the first protected monomer loaded therein. Thewash fluid can be forced into or drawn through and away from the one ormore reaction sites by a pumping block or pump connected to the firstmanifold, the second manifold, or both manifolds.

In a subsequent step, according to various embodiments, a deprotectingagent, for example, trichloroacetic acid, or the like, can be introducedinto the first manifold and the one or more surface tension controlvalves can be opened, enabling the deprotecting agent to pass throughand enter the one or more reaction sites. The deprotecting agent can bemoved into the one or more reaction sites by using positive pressure,negative pressure, gravity, centrifugal force, capillary action, or thelike. By contacting the first extended structure with the deprotectingagent in the one or more reaction sites, a deprotected extendedstructure can be formed in the one or more reaction sites. Excessdeprotecting fluid can then be forced out or drawn out of the one ormore reaction sites and through the second manifold, for example, byusing a pumping block to create a negative pressure differential. At thesame time, or subsequently, a wash fluid from the first manifold orsecond manifold can be forced into or drawn through and away from theone or more reaction sites that had the deprotecting agent loadedtherein. The wash fluid can be forced into or drawn through and awayfrom the one or more reaction sites by a pumping block or pump connectedto the first manifold, the second manifold, or both manifolds.

The wash fluid can then be removed from the reaction site by a pumpingblock, by air pressure, by centrifugal force, or the like. According tovarious embodiments, the deprotecting agent and the wash fluid can beremoved together from one or more of the reaction sites.

After removing the deprotecting agent, a second protected monomer canthen be introduced from the first manifold, through the one or moresurface tension control valves, and into one or more of the reactionsites. The second monomer can then bond to the deprotected extendedstructure, if present in the respective reaction site, to thereby form asecond extended oligomer structure having at least two monomericsubunits.

The abovementioned method can be repeated multiple times until a desiredoligomer has been formed. Once a completed oligomer has been formed, itcan be cleaved from its attachment site in the reaction site andcollected, for example, through the first or second manifold.

According to various embodiments of the method, and with reference tothe device shown in FIG. 5, the first manifold 503 can be used fordelivering reagents into the plurality of reaction sites, while thesecond manifold 517 can be used for drawing out or purging excessreagents from the reaction sites. Alternatively or additionally, thesecond manifold can be used to deliver reagents or wash fluid into oneor more of the reaction sites 510. Each surface tension control valvecan be controlled independently so a user can independently selectwhether a particular reagent is able to enter a reaction site from thefirst manifold 503. In this way, a different oligomer can be produced ineach reaction site. In oligonucleotide synthesis, this selectivesynthesis can be accomplished by the selective introduction of monomers,deprotecting agent, washing fluid, or a combination thereof.

According to various embodiments of the method, the fluid processingdevice can be used to synthesize at least two different oligonucleotideprimers and an oligonucleotide probe (as described above) in threedifferent interconnected reaction regions, for example, in the threedifferent reaction sites of the device shown in FIG. 6. In an exemplaryembodiment, one primer can be formed in reaction site 614, a secondprimer can be formed in reaction site 618, and a probe can be formed orpreloaded into reaction site 616. After formation of the primers, theycan be cleaved from their respective reaction sites and combined intothe reaction site 616 containing the probe. A nucleic acid sample canthen be introduced into the reaction site 616 along with suitablereagents for a PCR reaction. The reaction site 616 can then be sealedwith oil, a polymer, or with mechanical valving, to prevent evaporation,and then the contents of the site can be thermally cycled. In suchembodiments, the device can be used for reagent synthesis and PCR usingthe reagent.

All references, patents, patent applications, and patent applicationpublications cited herein are incorporated in their entireties byreference for all purposes.

Those skilled in the art can appreciate from the foregoing descriptionthat the present teachings can be implemented in a variety of forms.Therefore, while these teachings have been described in connection withembodiments thereof, the teachings should not be so limited. Variouschanges and modifications can be made without departing from theteachings herein.

1. A fluid processing device comprising: a plurality of reaction sites;a first fluid transport manifold in fluid communication with each of theplurality of the reaction sites; a second fluid transport manifold influid communication with each of the plurality of sites; and a pluralityof surface tension control valves, at least one of the plurality ofsurface tension control valves disposed between the first manifold andat least one respective reaction site of the plurality of reactionsites, each surface tension control valve being in fluid communicationwith the first manifold and the at least one respective reaction site.2. The fluid processing device of claim 1, wherein the first manifoldcontains one or more nucleic acid base selected from adenine, cytosine,guanine, and thymine.
 3. The fluid device of claim 1, further comprisinga dimethyltrityl-protected phosphoramidite nucleotide monomer disposedin the first manifold.
 4. The fluid processing device of claim 1,further comprising a planar substrate, wherein the first manifold, thesecond manifold, and the plurality of reaction sites are formed in thesubstrate.
 5. The fluid processing device of claim 1, wherein at leastone of the plurality of surface tension control valves comprises alight-actuated valve.
 6. The fluid processing device of claim 1, whereinat least one of the plurality of surface tension control valvescomprises an electrically-actuated valve.
 7. The fluid processing deviceof claim 1, wherein at least one of the plurality of surface tensioncontrol valves comprises a temperature-actuated valve.
 8. The fluidprocessing device of claim 1, further comprising a fluid communicationdirectly between two adjacent reaction sites of the plurality ofreaction sites.
 9. A system comprising the fluid processing device ofclaim 1, a plurality of respective different sources of nucleic acidbases, and a loading device for individually loading the differentnucleic acid bases from the plurality of respective different sourcesinto the first manifold.
 10. A system including the fluid processingdevice of claim 1, and a pressure differential source in fluidcommunication with one or more of the first manifold and the secondmanifold.
 11. A system comprising the fluid processing device of claim5, and an electromagnetic radiation source adapted to emitelectromagnetic radiation toward one or more of the plurality of surfacetension control valves.
 12. The system of claim 11, wherein theelectromagnetic radiation source includes a laser.
 13. The system ofclaim 11, further comprising a reflective device adapted to reflectelectromagnetic radiation emitted from the electromagnetic radiationsource toward one or more of the plurality of surface tension controlvalves.
 14. The system of claim 13, wherein the reflective devicecomprises a plurality of individual moveable mirrors.
 15. The system ofclaim 11, further comprising a control unit operatively connected to theelectromagnetic radiation source and adapted to control theelectromagnetic radiation source.
 16. The system of claim 11, furthercomprising at least one focusing lens disposed along an emission beampath between the electromagnetic radiation source and at least one ofthe plurality of surface tension control valves.
 17. The system of claim1, wherein the fluid processing device comprises at least one fluidcommunication between at least two of the plurality of reaction sites,and the at least one fluid communication bypasses the first and secondmanifolds.
 18. A system comprising the fluid processing device of claim1, and a thermal cycling block adapted to hold the fluid processingdevice such that at least one of the plurality of reaction sites is inheat-transfer communication with the thermal cycling block.
 19. A systemcomprising the fluid processing device of claim 1, and a rotatableplaten comprising a top surface, and a holder adapted to hold the fluidprocessing device in or on the top surface.
 20. A system comprising thefluid processing device of claim 1, and a pump adapted to connect to thefirst manifold and force liquid into the first manifold.
 21. A systemcomprising the fluid processing device of claim 6, and an electricitysource electrically connected to the electrically-actuated valve. 22.The system of claim 21, further comprising a control unit operativelyconnected to the electricity source and adapted to control theelectricity source.
 23. A system comprising the fluid processing deviceof claim 7, and a heater in heat-transfer communication with thetemperature-actuated valve.
 24. The system of claim 23, furthercomprising a control unit operatively connected to the heater andadapted to control the heater.
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)32. (canceled)
 33. (canceled)
 34. (canceled)